Methods of treating diseases associated with high-fat diet and vitamin a deficiency using retinoic acid receptor agonists

ABSTRACT

This invention relates to pharmaceutical composition and methods of using vitamin A and/or RARβ agonist for the treatment or prevention of diseases or conditions associated with high fat diet and/or vitamin deficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/754,438, filed Jan. 18, 2013 which is incorporated herein in itsentirety.

GOVERNMENT FUNDING

This invention was made with Government support under Grant Number R01CA043796 awarded by National Institutes of Health (NCI) and Grant NumberR01 DE10389 from the Dental and Craniofacial Institute. The UnitedStates Government has certain rights in the invention.

FIELD

The invention relates to the treatment and prevention of variousdiseases or conditions caused by fat accumulation or vitamin deficiency.

BACKGROUND

After smoking, high fat diet is said to be the second most lethal habit,causing 300,000 deaths each year in the U.S. alone. High fat diet leadsto many health problems, including obesity, stroke, cancer, high bloodpressure, diabetes, osteoarthritis, rheumatoid arthritis, multiplesclerosis, heart disease, and diseases in other organs such as liver andkidney.

Diabetes is a group of pancreatic diseases characterized by high bloodglucose levels that result from defects in the body's ability to produceand/or use insulin. In 2011 there were an estimated 366 million cases ofdiabetes worldwide, according to the International Diabetes Federation,and these cases are estimated to increase to 522 million by 2030 (1, 2).In the U.S. there were 23.7 million diagnosed cases, with an estimatedhealthcare cost of $113 billion (2, 3). Diabetes results when insulinproduction by pancreatic β-cells does not meet the metabolic demand ofperipheral tissues such as liver, fat, and muscle (4). A reduction inβ-cell number and function leads to hyperglycemia in both type I andtype II diabetes (4), in type I diabetes, insulin-producing pancreaticβ-cells lose self-tolerance and this gives rise to hyperglycemia (5).Each year in the United States there are over 30,000 new cases of type Idiabetes diagnosed (6). Patients with type I diabetes can control theirblood glucose level with insulin supplements (7). However, thedifferentiation of stem cells into pancreatic β-cells could be a longterm, better solution (8-10).

Type II diabetes is more common. In type II diabetes the body does notuse insulin properly, thus it is called insulin resistance. At first,the pancreas may make extra insulin to make up for it. But over timethere won't be enough insulin to keep blood glucose at normal levels.Type II diabetes is an increasingly prevalent disease that due to a highfrequency of complications leads to a significant reduction of lifeexpectancy. Because of diabetes associated microvascular complications,type II diabetes is currently the most frequent cause of adult-onsetloss of vision, renal failure, and amputations in the industrializedworld. In addition, the presence of type II diabetes is associated witha two to five fold increase in cardiovascular disease risk. After longduration of disease, most patients with type II diabetes will eventuallyfail on oral therapy and become insulin dependent with the necessity fordaily injections and multiple daily glucose measurements.

A third type of diabetes, gestational diabetes, is developed by manywomen usually around the 24th week of pregnancy. Treatment forgestational diabetes aims to keep blood glucose levels equal to those ofpregnant women who don't have gestational diabetes.

Some patients with diabetes can manage their conditions with healthyeating and exercise. Some will need to have prescribed medicationsand/or insulin to keep blood glucose levels. In addition, diabetes is aprogressive disease. Even if medication is not required at first, it maybe needed overtime.

Non-alcoholic fatty liver disease (NAFLD) is marked by lipidaccumulation in hepatocytes (steatosis) without evidence of hepatitis orliver fibrosis (69, 70). NAFLD is a major risk factor for development ofnon-alcohol steatohepatitis (NASH) and hepatocellular carcinoma (71).Driven by rising rates of obesity, diabetes and insulin resistance,NAFLD is currently the most common form of liver disease in the UnitedStates with an estimated 55 million cases (69). At the current rate,NAFLD will reach epidemic proportions in the United States by 2030; yetno FDA approved pharmacological therapy exist for prevention ortreatment of NAFLD (69).

Over the last decade, experimental animal and human data suggests thathepatic stellate cells (HSCs) are an important cellular target fordevelopment of pharmacological therapies for prevention or treatment ofNAFLD spectrum liver diseases (73). HSCs are star-like cells that residein the liver sinusoids whose main function are to store 80-90% of thetotal body vitamin A (VA) pool (74). During hepatic injury HSCs losingtheir VA storage capacity, trans-differentiate into myofibroblasts andorchestrate wound healing by secreting components of extra-cellularmatrix including type 1 collagen (col1a1) and alpha-smooth muscle actin(α-SMA) (72, 73). During pathogenesis of unchecked NAFLD, HSCsproliferate and become highly fibrotic through hyper-secretion col1a1and α-SMA leading to liver scarring and an inflammation cascade thatdrives further hepatic fibrosis and liver damage (72,73).

Diabetes is the most common cause of kidney failure, accounting fornearly 44 percent of new cases. Even when diabetes is controlled, thedisease can lead to Chronic Kidney Disease (CKD) and kidney failure.Nearly 24 million people in the United States have diabetes, and nearly180,000 people are living with kidney failure as a result of diabetes.People with kidney failure undergo either dialysis, an artificialblood-cleaning process, or transplantation to receive a healthy kidneyfrom a donor. In 2005, care for patients with kidney failure cost theUnited States nearly $32 billion.

There is an unmet medical need for methods, medicaments andpharmaceutical compositions with regard to disease-modifying propertiesand with regard to reduction of high fat diet or vitamin A deficiencyassociated diseases while at the same time showing a good safetyprofile.

SUMMARY

This invention discloses pharmaceutical compositions and methods fortreating and preventing diseases in pancreas, liver, kidney, testes, aswell as other organs that are associated with high fat diet and/orvitamin A deficiency.

According to certain embodiments, the invention provides a method oftreating or preventing a pancreatic disease in a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor:beta (RARβ).

In certain embodiments, the pancreatic disease is associated withobesity.

In certain embodiments, the pancreatic disease is associated with a highfat diet.

In certain embodiments, the pancreatic disease is associated withvitamin A deficiency in the pancreas.

The pancreatic disease may be diabetes, which may be type I or type IIdiabetes, or gestational diabetes.

According to certain embodiments, the invention provides a method ofincreasing RARβ level in a subject comprising administering to thesubject vitamin A or an agonist of retinoic acid receptor-beta (RARβ).

In certain embodiments, RARβ level is increased in an organ.

The organ may be pancreas, liver, kidney, or testes.

According to certain embodiments, the invention provides a method oftreating or preventing the degeneration of pancreatic beta cells in asubject comprising administering to the subject vitamin A or an agonistof retinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofmaintaining or improving the function of pancreatic beta cells in asubject comprising administering to the subject vitamin A or an agonistof retinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofmaintaining or improving pancreatic insulin secretion in a subjectcomprising administering to the subject vitamin A or an agonist ofretinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofmaintaining or improving insulin sensitivity in a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofmaintaining or improving the level of glucagon in a subject comprisingadministering to said subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).

According to certain embodiments, the invention provides a method oftreating or preventing fat deposit of a subject comprising administeringto the subject vitamin A or an agonist of retinoic acid receptor-beta(RARβ).

According to certain embodiments, the invention provides a method oftreating or preventing inflammation of a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofdecreasing the level of an inflammatory mediator in a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofdecreasing oxidative stress in a subject comprising administering to thesubject vitamin A or an agonist of retinoic acid receptor-beta (RARβ).

In certain embodiments, the production of the inflammatory mediator isdecreased.

In certain embodiments, the secretion of the inflammatory mediator isdecreased.

The inflammatory mediator may be monocyte chemotactic protein (mcp-1) ortumor necrosis factor alpha (tnf-α) according to certain embodiments.

In certain embodiments, the fat deposit, inflammation or oxidativestress is in an organ.

The organ may be pancreas, liver, kidney, or testes.

According to certain embodiments, the invention provides a method oftreating or preventing a liver disease in a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).

In certain embodiments, the liver disease is associated with obesity.

In certain embodiments, the liver disease is associated with a high fatdiet.

In certain embodiments, the liver disease is associated with vitamin Adeficiency.

In certain embodiments, the liver disease is fatty liver disease (FLD),liver fibrosis, or hepatic steatosis.

In certain embodiments, the liver disease is non-alcoholic FLD (NAFLD),alcohol associated FLD, or non-alcoholic steatohepatitis (NASH).

In certain embodiments, the liver disease is associated with reducedvitamin A level in the liver.

According to certain embodiments, the invention provides a method ofdecreasing the activation of hepatic stellate cells (HSCs) in a subjectcomprising administering to the subject vitamin A or an agonist ofretinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofdecreasing the level of hepatic reactive oxygen species (ROS) in asubject comprising administering to the subject vitamin A or an agonistof retinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofdecreasing the level of alpha smooth muscle actin (α-SMA) in a subjectcomprising administering to the subject vitamin A or an agonist ofretinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofincreasing the level of lethicin:retinol acyltransferase (LRAT) in theliver of a subject comprising administering to the subject vitamin A oran agonist of retinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofincreasing the level of RARβ in the liver of a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofdecreasing the level of SRBP1c in the liver of a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).

In certain embodiments, the subject has a liver disease.

In certain embodiments, the liver disease is fatty liver disease (FLD),liver fibrosis, or hepatic steatosis.

In certain embodiments, the liver disease is non-alcoholic FLD (NAFLD),alcohol associated FLD, or non-alcoholic steatohepatitis (NASH).

In certain embodiments, the liver disease is associated with reducedvitamin A level in the liver.

In certain embodiments, the liver disease is associated with a pancreasdisease.

According to certain embodiments, the invention provides a method oftreating or preventing a kidney disease in a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).

In certain embodiments, the kidney disease is associated with obesity.

In certain embodiments, the kidney disease is associated with a high fatdiet.

In certain embodiments, the kidney disease is kidney fibrosis.

In certain embodiments, the kidney disease is a chronic kidney disease.

In certain embodiments, the kidney disease is associated with apancreatic disease.

In certain embodiments, the kidney disease is associated with a liverdisease.

In certain embodiments, the kidney disease is associated with reducedvitamin A level in the kidney.

According to certain embodiments, the invention provides a method ofincreasing the level of lethicin:retinol acyltransferase (LRAT) in thekidney of a subject comprising administering to the subject vitamin A oran agonist of retinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method oftreating or preventing a disease associated with an organ-specificvitamin A deficiency in a subject comprising administering to thesubject vitamin A or an agonist of retinoic acid receptor-beta (RARβ).

In certain embodiments, the organ-specific vitamin A deficiency isassociated with obesity.

In certain embodiments, the organ-specific vitamin A deficiency isassociated with a high fat diet.

In certain embodiments, the subject has a normal serum level of vitaminA or retinyl esters.

The organ may be pancreas, liver, or kidney.

According to certain embodiments, the invention provides a method oftreating or preventing fibrosis in a subject comprising administering tothe subject an agonist of retinoic acid receptor-beta (RARβ).

According to certain embodiments, the invention provides a method ofdecreasing the accumulation of fat in a subject comprising administeringto the subject an agonist of retinoic acid receptor-beta (RARβ).

In certain embodiments, the fibrosis or accumulation of fat is in anorgan.

The organ may pancreas, liver, kidney, or testes.

According to certain embodiments, the vitamin A or agonist of retinoicacid receptor-beta (RARβ) is administered three times daily.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) is administered at an amount from 30-200 mg perday.

In certain embodiments, the vitamin A or agonist is administered at anamount from 50-150 mg per day.

In certain embodiments, the vitamin A or agonist is administered at anamount from 50-100 mg per day.

In certain embodiments, the vitamin A or agonist is administered at anamount from 100-150 mg per day.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) is administered orally.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) is administered intravenously or subcutaneously.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) does not elevate serum triglyceride in the subject.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) does not increase cardiovascular risk in thesubject.

In certain embodiments, a therapeutic effective amount of the vitamin Aor agonist of RARβ is administered.

In certain embodiments, both vitamin A and an agonist of RARβ are bothadministered to the subject.

In certain embodiments, vitamin A and an agonist of RARβ areadministered concomitantly.

In certain embodiments, vitamin A and an agonist of RARβ areadministered sequentially.

According to certain embodiments, the invention provides apharmaceutical composition comprising vitamin A or an agonist ofretinoic acid receptor-beta (RARβ) or a pharmaceutically acceptable saltthereof at an amount from about 10 mg to about 60 mg.

In certain embodiments, the amount of the vitamin A or agonist is from15 mg to about 50 mg.

In certain embodiments, the amount of the vitamin A or agonist is from15 mg to about 35 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 35 mg to about 50 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 30 mg to about 200 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 50 mg to about 150 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 50 mg to about 100 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 100 mg to about 150 mg.

According to certain embodiments, the invention provides apharmaceutical composition comprising vitamin A or an agonist ofretinoic acid receptor-beta (RARβ) or a pharmaceutically acceptable saltthereof at a concentration from about 0.1 mg to about 10 mg per 100 ml.

In certain embodiments, the concentration is from about 0.5 mg to about5 mg per 100 ml.

In certain embodiments, the concentration is from about 1 mg to about2.5 mg per 100 ml.

In certain embodiments, the agonist is a highly specific RARβ agonist.

In certain embodiments, the agonist is AC261066.

In certain embodiments, the agonist is AC55649.

In certain embodiments, the pharmaceutical composition comprises bothvitamin A and an agonist of RARβ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Pancreatic endocrine differentiation protocol and its impact onthe molecular level. (A) Schematic representation of the endocrinedifferentiation protocol adapted from D'Amour et al. (2006) used onmouse ES cells. Briefly, embryonic stem (ES) cells are treated withdifferent growth factors to successively differentiate into definitiveendoderm (DE), pancreatic progenitor (PP), endocrine progenitor (EP),and endocrine cells (EC). (B) WT mouse ES cells were subjected to the17-day differentiation protocol. Each lane represents a differentcondition at specific time points. RT-PCR analyses were performed tomonitor the expression of pancreatic differentiation markers such asinsulin-1 (Ins1), glucagon (Gcg), somatostatin (Sst), neurogenin-3(Ngn3), Pdx1 and Sox17, as well as the stem cell markers Nanog and Rcx1.HPRT1 amplification was used as a control housekeeping gene. Pancreasextracts from CS7BL/6 WT mice were used as a positive control.

FIG. 2: Impact of RARβ deletion on Pdx1 expression through pancreaticendocrine differentiation process. (A) RT-PCR analysis confirming thesuppression of RARβ in KO ES cells. Analysis of Cyp26a1, a RA-responsivegene, demonstrates the presence of RA signaling activity via otherreceptors in RARβ KO cells. HPRT1 was used as a control housekeepinggene. (B) Indirect immunofluorescence staining for Pdx1 (green) in WTand RARβ KO, at 5, 11, 14, and 17 days in the absence (untreated) or inthe presence (treated) of growth factors used in the differentiationprotocol. Cells were counterstained using rhodamine-conjugatedphalloidin, which binds to F-actin (red) and nuclei were stained withDAPI (blue) (Bars=50 μm).

FIG. 3: Expression of pancreatic differentiation markers in WT and RARβknockout (KO) ES cells. Transcript expression analyses of (A) early, (B)mid, and (C) late stage endocrine pancreatic differentiation markers inWT and RARβ KO ES cells. RT-PCR amplification of (A) Nanog, Ngn3, (B)Pax6, Is1-1, and (C) Ins1, Gcg, and Iapp mRNA was performed in both celllines at 5, 11, 14, and 17 days of the differentiation protocol. In eachcase, RARβ expression was monitored in both cell lines and HPRT1 wasused as a control housekeeping gene. Relative amounts, normalized toHPRT1 levels for each marker tested, are shown in histograms (n=3; *:p≦1,05; **: p≦0.0079; ***: p≦0.0003).

FIG. 4: In vivo characterization of RARβ deletion on islets ofLangerhans functionality and glucose metabolism. (A) Indirectimmunofluorescence staining of C-peptide (green) and Glucagon (red) onC57BL/6 WT and RARβ KO mouse pancreas tissue sections. Pancreaticislet-corresponding regions were circled by dashed lines and nuclei weremarked with DAPI (blue) (bars=50 μm). Islet size were quantified persurface area units (cm²), with respect to high resolution micrographs,for each group and presented as histogram (n=6; **: p=0.031), Westernblot analysis of C-peptide and Glucagon expression was performed on WTand RARβ KO mouse pancreas protein extracts. Ins-1 cells were used aspositive control for C-peptide expression, while immunodetection ofactin was used as a loading control. (B) Blood glucose concentration(mg/dL) in WT and RARβ null, knockout mice after 15 h fasting (left)(n≧5; p=0.0011). Blood glucose clearance (right) for WT (♦) and RARβ KO(▪) mice was measured following a 2 mg/kg dextrose i.p. injection.Relative blood glucose levels were assessed at 0, 15, 30, 45, 60, and120 minutes post-injection (n≧6; *: p=0.0137; ***: p<0.0001).

FIG. 5. Retinoid levels in mouse pancreas following the treatmentindicated. Con fed diet (CFD) (n=5); HFD (n=5). Mice fed a high fatdiet/obese mice have almost no retinoids in the pancreas compared tomice on a control, normal chow diet. They exhibit an organ specificvitamin A deficiency.

FIG. 6. Serum retinol from mice on a high fat diet vs. control dietcompared to the pancreas retinol and retinyl palmitate levels from miceon a high fat vs. control diet. The serum retinol levels are similar ora bit higher in the HF diet mice, but the pancreas retinol levels aremuch lower in the HF diet mice, showing vitamin A deficiency in thepancreas even in the presence of normal serum vitamin A.

FIG. 7. 4-hydroxynonenal (4-HNE), an indicator of oxidative stress, inthe pancreas. The pancreas samples were fixed, embedded in paraffin, andsectioned. Then the tissue sections were stained with an antibodyagainst 4-HNE (magnification, 200×). Sections from two mice/group werephotographed and analyzed. The arrows indicate the pancreatic islets.AC261066 reduces oxidative stress in the pancreatic islets in mice on ahigh fat diet (HFD+AC261066).

FIG. 8. AC261066 slightly reduces expression of c-peptide (marker ofinsulin secretion stress) in islets of HF fed mice. Representativeimmunofluorescence stained pancreatic sections front wild type (wt) maleC57/BL6 mice fed either chow control diet (Con), high fat (HF) diet, HFdiet plus AC261066 for 4 months. Con Diet (n=5); HF diet (n=5); HFDiet+AC261066 (n=5). Blue, nuclei of cells; red, glucagon; green,c-peptide.

FIG. 9. Gene expression of INS2, RARB2, CYP26A1 and LRAT in pancreatictissue from wild type (wt) male C57/BL6 mice fed either chow controldiet (Con), high fat (HF) diet, HF diet plus AC261066, Cyp26 and LRAT,no detectable signal. HPRT, loading control. RAR β2 mRNA levels weredecreased by the high fat diet compared to the control diet (con),consistent with the vitamin A deficiency in the pancreas. AC261066increased the RAR β2 mRNA levels in the HF diet mice.

FIG. 10. Gene expression of RARβ2 in pancreatic tissue from LRAT −/−vitamin A sufficient mice (VAS, normal control diet), LRAT −/− vitamin Adeficient (VAD) mice, and LRAT −/− vitamin A deficient (VAD) micetreated with AC261066 for S weeks. AC261066 increased the RARβ2 mRNA,levels in vitamin A deficient mice (LRAT −/− on a VAD diet for 4 months.

FIG. 11, AC261066 diminished hepatic steatosis. Representativehematoxylin and eosin stained liver sections from wild type (wt) maleC57/BL6 mice fed either a chow control diet (Con), high fat (HF) diet,HF diet plus AC261066 or HF diet plus CD1530 (RAR gamma agonist) for 4months. Con Diet (n=5); HT diet (n=5); HF Diet+AC261066 (n=5), or HFdiet+CD1530 (RAR gamma agonist) (n=4).

FIG. 12. Gene Expression in Livers of Control and HF-Fed Mice. Geneexpression of SREBP1c and α-SMA in livers from wild type (wt) maleC57/BL6 mice fed either a chow control diet (Con), high fat (HF) diet, aHF diet plus AC261066 or HF diet plus CD1530 (RAR gamma agonist) for 4months. Con Diet (n=5); HF diet (n=5); HF Diet+AC261066 (n=5), or HFdiet+CD1530 (RAR gamma agonist) (n=4).

FIG. 13. AC261066 diminished activation of hepatic stellate cells.Representative immunofluorescence and oil red o stained liver sectionsfrom wild type (wt) male C57/BL6 mice fed either a chow control diet(Con), high fat (HF) diet, HF diet plus AC261066 or HF diet plus CD1530(RAR gamma agonist) for 4 months. Control Diet (n=5); HF diet (n=5); HFDiet+AC261066 (n=5), or HF diet+CD1530 (RAR gamma agonist).

FIG. 14. Gene Expression of inflammatory Mediators in Livers of LF andHF-Fed Mice. Gene expression of MCP-1, TNF-alpha in livers from wildtype (wt) male C57/BL6 mice fed either a chow control diet (Con), highfat (HF) diet, HF diet plus AC261066 or HF diet plus CD1530 (RAR gammaagonist) for 4, LF Diet (n=5); HF diet (n=5); HF Diet+AC261066 (n=5), orHF diet+CD1530 (RAR gamma agonist) (n=4). AC261066 decreases levels ofinflammatory proteins MCP-1 and TNF alpha in livers of HF diet fed mice.

FIG. 15. Mouse serum triglyceride levels following the treatmentsindicated. Con diet (n=2); HFD (n=3); HFDAC (n=5). Con, control diet;HFD, high fat diet; HFD+AC, high fat diet+AC261066. AC261066 does notincrease triglyceride levels at doses used.

FIG. 16. Retinoid levels in mouse liver following the treatmentsindicated. Con fed diet (CFD) (n=5); HFD (n=5); HFD+AC261066 (n=5),HFD+CD1530 (n=4). High fat diet caused a state of vitamin A deficiencyin liver and this is partially reversed by AC261066. Note that they-axes in the left panel are different for CFD and HFD. The HFD reducedretinyl esters, (retinyl palmitate), a form of storage of vitamin A inthe liver, by greater than 90% (left panel). The HFD also reducedretinol (vitamin A) levels by over 90% to result in vitamin A deficiencyin the liver.

FIG. 17. 4-hydroxynonenal (4-HNE), an indicator of oxidative stress, inthe liver. The liver samples were fixed, embedded in paraffin, andsectioned. Then the tissue sections were stained with an antibodyagainst 4-HNE (magnification, 200×). Sections from two mice/group werephotographed and analyzed. These data show that AC261066 reducesoxidative stress and ROS (reactive oxygen species) in the livers of HFdiet fed mice. Oxidative stress damages tissues.

FIG. 18. AC261066 diminished renal lipid accumulation. Representativehematoxylin and eosin stained kidney sections from wild type (wt) maleC57/BL6 mice fed either a chow control diet (Con), high fat gin diet, HFdiet plus AC261066 or HF diet plus CD1530 (RAR gamma agonist) for 4months. Con Diet (n=5); HF diet (n=5); HF Diet+AC261066 (n=5), or HFdiet+CD1530 (RAR gamma agonist) (n=4).

FIG. 19. AC261066 diminished expression of the fibrogenic proteinalpha-SMA. Representative immunofluorescence and oil red o stainedkidney sections from wild type (wt) male C57/BL6 mice fed either a chowcontrol diet (Con), high fat (HF) diet, or HF diet plus AC261066 for 4months. Chow Diet (n=5); HF diet (n=5); HF Diet+AC261066 (n=5), or HFdiet+CD1530 (RAR gamma agonist).

FIG. 20. Retinoid levels in mouse kidney following the treatmentsindicated. Con fed diet (CFD) (Lean) (n=5) or HFD (Obese)(n=5). The highfat diet led to dramatic declines in retinyl esters (retinyl palmitate)and retinol in the kidney, showing a vitamin A deficiency in kidney.

FIG. 21. Gene Expression of Inflammatory Mediators in Kidneys of ControlNormal Chow (13% fat) and HF—Fed Mice and RARs. Gene expression ofkidney from wild type (wt) male C57/BL6 mice fed either a chow controldiet (Con), high fat (HF) diet, HF diet plus AC261066. AC261066 reducesthe levels of TNF-alpha, a potent inflammatory protein, mRNA in high fatdiet fed mice. AC261066 also restores RAR beta and LRAT mRNA levels,markers of functional vitamin A signaling, in the high fat diet fedmice, 4 months on the HFD. HPRT, loading control.

FIG. 22. 4-hydroxynonenal (4-HNE), an indicator of oxidative stress, inthe kidneys. The kidney samples were fixed, embedded in paraffin, andsectioned. Then the tissue sections were stained with an antibodyagainst 4-HNE (magnification, 200×). Sections from two mice/group werephotographed and analyzed. AC261066 reduces oxidative stress (ROS) inthe kidneys of mice fed the HF diet.

FIG. 23. Retinoid levels in mouse testes following the treatmentsindicated. Con fed diet (CFD) (n=5) or HFD (n=5). High fat diet resultsin partial vitamin A deficiency in the testes.

FIG. 24. Gene Expression of vitamin A relevant genes in testes of Chowand HF-Fed Mice Gene expression of testes from wild type (wt) maleC57/BL6 mice fed either a chow control diet (Con), high fat (HF) diet,HF diet plus AC261066. (Each number is data from one mouse, five micetotal in each group.)

DETAILED DESCRIPTION

As discussed above, there remains a need to provide alternate therapiesor management for a variety diseases associated with high fat diet andvitamin A deficiency. Accordingly, the present invention relates to usesof vitamin A and retinoic acid receptor β (RARβ) agonists in thisregard.

Mouse embryonic stem (ES) cells are pluripotent cells derived from theinner cell mass of blastocyst-stage (day 3.5) embryos (10, 11). Upon LIFremoval, ES cells spontaneously differentiate into all three primaryembryonic germ layers: endoderm, mesoderm, and ectoderm (10). Severalresearch groups have shown that the directed differentiation of ES cellsalong the endocrine pathway can be achieved by using a wide range ofgrowth/differentiation factors, including retinoic acid (RA) treatment(12-17).

Although the effects of RA on cells and tissues are known to occurthrough the activation of retinoic acid receptors (RARα, RARβ, and RARγ)and their isoforms (6, 18), the events occurring downstream of RAsignaling that direct the differentiation of definitive endoderm intoendocrine precursors are poorly understood (4, 5, 19).

A series of in vivo experiments, including some in Xenopus revealed,however, that RA signaling is crucial for endocrine pancreaticdevelopment (20). For instance, mice containing an inducible transgenefor the dominant negative RARα403 mutant, used to ablate retinoicacid-dependent processes in vivo, lacked both a dorsal and ventralpancreas, and died at the neonatal stage (21). Impaired pancreatic isletdevelopment and repletion were also observed in vivo, in vitamin Adeficiency models (22, 23). Moreover, a study of the developmentalpathways involved during in vitro islet neogenesis revealed a 3-foldinduction of RARβ transcripts from ““adherent”” to ““expanded”” stagesof endocrine differentiation (24). Another study, based on the role ofCRABP1 and RBP4 in pancreatic differentiation, corroborated theup-regulation of RARβ in early differentiation (11). While previousstudies suggested that RARβ is essential to pancreas development, littleis known about its functional role in pancreas formation and isletmaintenance in adults (25, 26).

Vitamin A metabolite all trans-retinoic acid (RA) acting through itscognate receptors, retinoic acid receptor (RAR) alpha, beta, gamma,possesses anti-obesity and anti-lipogenic properties through regulationof genes involved in energy metabolism and adipogenesis (75).

Using animal models, the present inventors have discovered that retinoicacid receptor β (RARβ) plays an important role in organ development,maintenance, and function. The inventors discovered that vitamin A andRARβ agonists increase RARβ function and signaling; vitamin A and theseRARβ agonists also increase the level of RARβ.

The present inventors also discovered that vitamin A and RARβ agonistsare effective in treating and preventing high at diet associated diseasein pancreas, liver, kidney, testes and other organs. Furthermore, theinventors discovered that vitamin A and such (RARβ) agonists can restorevitamin A signaling in organs that show vitamin A deficiencies.

Vitamin A and these RARβ agonists, according to the discovery of thepresent inventors, increase insulin signaling, decrease fat deposit,prevent inflammation, and decrease oxidative stress in various organs,including pancreas, liver, kidney and testes. They also decrease thelevel of alpha smooth muscle actin (α-SMA) but increase the level oflethicin:retinol acyltransferase (LRAT) and RARβ. When used to treatliver diseases, vitamin A and these RARβ agonists decrease theactivation of hepatic stellate cells (HSCs) and the level of hepaticreactive oxygen species (ROS).

The present inventors discovered that vitamin A or agonists of retinoicacid receptor-beta (RARβ) do not elevate serum triglyceride or increasecardiovascular risk at a clinically significant level.

The retinoic acid receptor (RAR) is a type of nuclear receptor that isactivated by both all-trans retinoic acid and 9-cis retinoic acid. Thereare three retinoic acid receptors (RAR), RARα, RARβ, and RARγ, encodedby the RARα, RARβ, RARγ genes, respectively. Each receptor isoform hasseveral splice variants: two- for α, four- for β, and two- for γ.

RAR heterodimerizes with RXR and in the absence of ligand, the RAR/RXRdimer binds to hormone response elements known as retinoic acid responseelements (RAREs) complexed with corepressor protein. Binding of agonistligands to RAR results in dissociation of corepressor and recruitment ofcoactivator protein that, in turn, promotes transcription of thedownstream target gene into mRNA and eventually protein.

Known RARβ agonists include but are not limited to: AC261066, AC55649,LEI35, Tazarotene, Adapalene, CD666, 9-cis-retinoic acid, BMS641 andTTNPB. AC261066 and AC55649 are highly-specific RARβ agonists. The term“highly-specific RARβ agonists” also include other agonists having abinding affinity similar to AC261066 or AC55649, e.g., at least 50% orgreater, preferably 75% or greater, more preferably 90% or greater ofthe RARβ binding affinity of AC261066 or AC55649.

RARβ agonists include the fluorinated alkoxythiazoles previouslydescribed (65), such as:

4′-Octyl-[1,1′-biphenyl]-4-carboxylic acid (65), Adapalene (67),BMS-231973, BMS-228987, BMS-276393, BMS-209641 (66), BMS-189453{4-[(1E)-2-(5,6-Dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]-benzoicacid} (68), CD2019(6-[4-methoxy-3-(1-methylcyclohexyl)phenyl]naphthalene-2-carboxylicacid), compounds described in WO20081064136 and WO2007009083 andtazarotene (ethyl6-[2-(4,4-dimethyl-3,4-dihydro-2H-1-benzothiopyran-6-yl)ethynyl]pyridine-3-carboxylate).

AC261066:

AC55649:

Tazarotene:

Adapalene:

CD666:

9-cis-retinoic acid:

BMS 641:

TTNPB:

The highly specific RARβ agonist, e.g., AC261066, can prevent hepaticsteatosis and activation of HSCs, marked by decreased expression ofα-SMA. AC261066 can significantly diminish hepatic gene expression ofpro-inflammatory mediators tumor necrosis factor-alpha (TNFα) andmonocyte chemotactic protein-1 (MCP-1).

As used herein, the term “subject” means an animal, preferably a mammal,and most preferably a human. A subject may be a patient having a diseaseor disorder as discussed herein.

As used herein, the term “vitamin A deficiency” refers to a lack ofvitamin A or a decreased level of vitamin in serum or an organ (e.g.,pancreas, liver, kidney or testes) of an animal, e.g., human.

As used herein, the terms “decreasing” and “reducing” are usedinterchangeably to refer to a negative change in the level, activity orfunction of a molecule, cell or organ. It is meant that the particularlevel, activity or function is lower by about 25%, about 50%, about 75%,about 90%, about 1-fold, about 2-fold, about 5 fold, about 10-fold,about 25-fold, about 50-fold, or about 100 fold, or lower, when comparedto a control.

As used herein, the terms “increasing”, “improving” and “enhancing” areused interchangeably to refer to a positive change in the level,activity or function of a molecule, cell or organ. It is meant that theparticular level, activity or function is higher by about 25%, about50%, about 75%, about 90%, about 1-fold, about 2-fold, about 5 fold,about 10-fold, about 25-fold, about 50-fold, or about 100 fold, orhigher, when compared to a control.

The expressions “therapeutically effective” and “therapeutic effect”refer to a benefit including, but not limited to, the treatment oramelioration of symptoms of a proliferative disorder discussed herein.It will be appreciated that the therapeutically effective amount or theamount of agent required to provide a therapeutic effect will varydepending upon the intended application (in vitro or in vivo), or thesubject and disease condition being treated (e.g., nature of theseverity of the condition to be treated, the particular inhibitor, theroute of administration and the age, weight, general health, andresponse of the individual patient), which can be readily determined bya person of skill in the art. For example, an amount of vitamin A or anagonist of RARβ is therapeutically effective if it is sufficient toeffect the treatment or amelioration of symptoms of a disease discussedherein.

The term “clinically significant level” is used herein to refer to alevel of a side effect such as cardiovascular risk caused by theadministration of a pharmaceutical composition (e.g., vitamin A or RARβagonist) that a physician treating the subject would consider to besignificant.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 30%, preferably 20%, more preferably 10%.

As used herein, the term “comprises” means “includes, but is not limitedto.”

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio.

If a pharmaceutically acceptable salt of vitamin A or agonist of RARβ isutilized in pharmaceutical compositions, the salt preferably is derivedfrom an inorganic or organic acid or base. For reviews of suitablesalts, see, e.g., Berge et al, J. Pharm. Sci. 66: 1-19 (1977) wadRemington: The Science and Practice of Pharmacy, 20th Ed., ed. A.Gennaro, Lippincott Williams & Wilkins, 2000.

The term “pharmaceutically acceptable carrier” is used herein to referto a material that is compatible with a recipient subject, preferably amammal, more preferably a human, and is suitable for delivering anactive agent to the target site without terminating the activity of theagent. The toxicity or adverse effects, if any, associated with thecarrier preferably are commensurate with a reasonable risk/benefit ratiofor the intended use of the active agent.

The term “carrier” is used interchangeably herein, and include any andall solvents, diluents, and other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington: TheScience and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, LippincottWilliams & Wilkins, 2000 discloses various carriers used in formulatingpharmaceutically acceptable compositions and known techniques for thepreparation thereof.

The pharmaceutical compositions of the invention can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders, tablets,capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions. Formulations may optionally contain solvents,diluents, and other liquid vehicles, dispersion or suspension aids,surface active agents, pH modifiers, isotonic agents, thickening oremulsifying agents, stabilizers and preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired.

The vitamin A or agonist of RARβ can be administered by any method knownto one skilled in the art. For example, vitamin A or agonist of RARβ maybe administered orally or parenterally.

The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally, intravenously, or subcutaneously. The formulations of theinvention may be designed to be short-acting, fast-releasing, orlong-acting. Still further, compounds can be administered in a localrather than systemic means, such as administration (e.g., by injection)at a tumor site.

Liquid dosage farms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents such as phosphates orcarbonates.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart.

Combination therapies that comprise the combination of vitamin A andagonist of RARβ of the present invention, and further with one or moreother therapeutic agents can be used, for example, to: 1) enhance thetherapeutic effect(s) of the methods of the present invention and/or theone or more other therapeutic agents; 2) reduce the side effectsexhibited by the methods of the present invention and/or the one or moreother therapeutic agents; and/or 3) reduce the effective dose of vitaminA or agonist of RARβ of the present invention and/or the one or moreother therapeutic agents.

The amount or suitable dosage of vitamin A or agonist of RARβ dependsupon a number of factors, including the nature of the severity of thecondition to be treated, the route of administration and the age,weight, general health, and response of the individual subject. Incertain embodiments, the suitable dose level is one that achieves thistherapeutic response and also minimizes any side effects associated withthe administration. For example, vitamin A or agonist of RARβ may beadministered at an amount from about 30 mg to about 200 mg per day,e.g., about 50 mg to about 150 mg per day, about 50 to about 100 mg perday, about 100 mg to about 150 mg per day.

Vitamin A or agonist of RARβ may be administered in single or divided ormultiple doses. It will be understood that a suitable dosage of vitaminA or agonist of RARβ may be taken at any time of the day or night, withfood or without food. In some embodiments, the treatment period duringwhich an agent is administered is then followed by a non-treatmentperiod of a particular time duration, during which the therapeuticagents are not administered to the patient. This non-treatment periodcan then be followed by a series of subsequent treatment andnon-treatment periods of the same or different frequencies for the sameor different lengths of time.

In the following description, reference is made to the accompanyingdrawings that form a part hereof, and in which is shown by way ofillustration specific embodiments which may be practiced. Theseembodiments are described in detail to enable those skilled in the artto practice the invention, and it is to be understood that otherembodiments may be utilized and that logical changes may be made withoutdeparting from the scope of the present invention. The followingdescription of example embodiments is, therefore, not to be taken in alimited sense, and the scope of the present invention is defined by theappended claims.

EXAMPLES

The present description is further illustrated by the followingexamples, which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication) are hereby expressly incorporated by reference.

Example 1 Materials and Methods

Cell Culture and Isolation of RARβ Homozygous ES Cell Line. Mouse J1wild-type ES cells were cultured as described previously (27). C57BL/6RARβ heterozygous mice were provided by Dr. Pierre Chambon(Strasbourg-Cedex, France) (26). Homozygous RARβ-null mice were obtainedfollowing mating of RARβ heterozygous mice. Blastocysts were harvestedon day E3.5 and individually cultured on ES cell medium as previouslydescribed (28).

Pancreatic endocrine differentiation protocol. A slightly modifiedversion of the established protocols published by Borowiak (14) andD'Amour (15) was used to carry out differentiation of hormone expressingendocrine cells from mouse ESCs. Prior to differentiation, ESCs wereseeded at 5×10⁵ on 30 mm gelatin-coated plates. After overnight culture,cells were exposed to 250 nM BIO-Acetoxime (EMD Bioscience, San Diego,Calif.)+50 ng/ml activin A (R&D Systems, Minneapolis, Minn.) in AdvancedRPMI (GIBCO, Grand Island, N.Y.) supplemented with 1× L-Glu and 0.2% FBS(GIBCO) for 1 day, and then to activin A alone in the same media. Cellswere then cultured for 4 days to induce endoderm differentiation. Forpancreatic progenitor induction, the cells were transferred to 50 ng/mlFGF10 (R&D Systems), 7.5 μM cyclopamine (Calbiochem, San Diego, Calif.)in DMEM supplemented with 1× L-Glu, 1× Pen/Strep, and 1× B27(Invitrogen, Grand Island, N.Y.) for 2 days. At day 7, cells weretransferred to FGF10, cyclopamine and 2 μM all-trans RA (Sigma, St.Louis, Mo.) in DMEM supplemented with 1× L-Glu, 1× Pen/Strep, and 1× B27(Invitrogen) for 4 days. At day 11, cells were cultured in the presenceof DMEM supplemented with 1× L-Glu, 1× Pen/Strep, and 1× B27 for 3 days.At day 14, CMRL (Invitrogen) medium was added and supplemented with 1×L-Glu, 1× Pen/Strep, 1× B27, 50 ng/ml IGF-1 (R&D Systems), 50 ng/ml HGF(R&D Systems), and 10 mM nicotinamide (Sigma) for 3 more days. All stockcompounds were made in either PBS or ethanol.

RT-PCR analysis. Various markers for endodermal (day 5), pancreaticprogenitor (day 11), endocrine progenitor (day 14) and endocrine (day17) differentiation were analyzed by semi-quantitative RT-PCR in J1wild-type and RARβ KO ESCs. Specific primers used and amplificationconditions are listed in Table-1. Primers were designed around intronswhenever possible. Primers not designed around introns are shown inTable 1 with an asterisk. Total RNA extraction, semi-quantitative andquantitative PCR reactions were performed as previously described (18).Amplified PCR products were resolved on 1.5% agarose gels and visualizedby staining with ethidium bromide. PCR bands were sequenced forverification of the correct amplicon. Quantitation of semi-quantitativegels was performed using Image) software (National Institutes of Health)from three experimental biological repeats.

TABLE 1 Primer sequences used for RT-PCRAll primers for RT-PCR are designed around introns, except those marked with *.Primer Application Forward sequence (5′→3′) Reverse sequence (5′→3′)Product size (bp) mlns1 RT-PCR GTGACCAGCTATAATCAGAG GCCAAGGTCTGAAGGTCC (SEQ 289 (SEQ ID No. 1) ID No. 2) mGcg RT-PCRGCCCGTGCCCAAGATTTT TGCGGCCGAGTTCCT 232 (SEQ ID No. 3) (SEQ ID No. 4)mSst* RT-PCR GCCCAACCAGACAGAGAA AGTTCTTGCAGCCAGCTT 150 (SEQ ID No. 5)(SEQ ID No. 6) mNgn3- RT-PCR TGCGCATAGCGGACCACAGCTTCCACAAGAAGTCTGAGAACACCAG 233 (SEQ ID No. 7) (SEQ ID No. 8) mRARβ RT-PCRATCCTGGATTTCTACACCG CTGACGCCATAGTGGTA 248 (SEQ ID No. 9) (SEQ ID No. 10)mNanog RT-PCR AGGATGAAGTGCAAGCGGTGG GATAAGACACCACAGTACACAC 520(SEQ ID No. 11) (SEQ ID No. 12) mRex1 RT-PCR AACATTGCAGATGGTGCTTCAGGGCTGAAGGCCTGCATAATCACC 641 (SEQ ID No. 13) (SEQ ID No. 14) mCyp26a1RT-PCR AACATTGCAGATGGTGCTTCAG GGCTGAAGGCCTGCATAATCAC 272 (SEQ ID No. 15)(SEQ ID No. 16) mPax-6 RT-PCR AACCCCCAGTCCCCAGTCAGAGTCCATTCCCGGGCTCCAGTTCA 399 (SEQ ID No. 17) (SEQ ID No. 18) misl-1*RT-PCR CGGGGGCCACTATTTG GGCACGCATCACGAA 397 (SEQ ID No. 19)(SEQ ID No. 20) miapp* RT-PCR GGCTGTAGTTCCTGAAGC ACTTCCGTTTGTCCATCT 199(SEQ ID No. 21) (SEQ ID No. 22) HPRT1 RT-PCR CTCGAGATGTGATGAAGGCCCTGTTGACTGGTCATT 192 (SEQ ID No. 23) (SEQ ID No. 24)

Indirect Immunofluorescence, Immunofluorescence assays on cells andtissue sections were performed as previously described (29). Briefly,differentiated samples were fixed using 4% (w/v) paraformaldehyde andmembrane permeabilization (for cells only) was done with 0.3% (w/v)Triton-X 100 (Sigma). Unspecific sites were blocked using 2% BSA for 30min prior to incubation with rabbit polyclonal anti-PDX1 (Millipore,06-1379, 1:1000), rabbit anti-C-Peptide (Cell Signaling, 4593, 1:500,Danvers, Mass.) and mouse monoclonal anti-Glucagon (Abcam, ab1.0988,1:200) primary antibodies. Phalloidin-TRITC (Millipore, FAK100, 1:1000,Billerica, Mass.) was used to stain the actin stress fiber network(F-actin). Nuclei were stained using DAPI contained in Vectashield®mounting medium for fluorescence (Vector labs, Burlingame, Calif.).Quantitation of C-peptide positive stained cells and islet surface areawas performed using NIS-Elements Advanced Research software (Nikon).

Western blot analysis. Proteins were extracted from mouse pancreas,separated by SDS-PAGE, and transferred onto nitrocellulose membranes aspreviously described (30, 31). Membranes were blocked in PBS containing5% skim milk and 0.1% TWEEN 20 (BioRad, Hercules, Calif.). Rabbitanti-C-Peptide (Cell Signaling, 4593, 1:500), mouse monoclonalanti-Glucagon (Abcam, ab10988, 1:500) and anti-actin (Millipore,MAB1501, 1:2000) primary antibodies were incubated with membranesovernight at 4° C.

Mouse Blood Glucose Assays, C57BL/6 WT and RARβ KO mice were used forthis experiment as previously described (26). Briefly, mice were fastedfor 15 hours overnight and a 50% dextrose solution (2 g/kg body weight)was injected intraperitoneally. Blood glucose levels were measured fromthe tail vein at 0, 15, 30, 60, and 120 min using the One Touch BloodGlucose Monitoring System (LifeScan) (32).

Statistical analysis. All experiments were performed at least 3 times(n>3) using independent biological triplicates. Results were presentedas means±SEM. All statistical tests were performed using GraphPad InStatsoftware version 3.10. A p-value of ≦0.05 indicated statisticalsignificance.

Example 2 Pancreatic Differentiation and Assessment of PancreaticMarkers in WT Mouse ES Cells

The endocrine differentiation protocol was selected because it includedRA-treatment at day 7 and also showed expression of later stageendocrine markers using human ES cells (15). The D'Amour et al. (2006)pancreatic differentiation protocol was used with some slightmodifications to generate pancreatic endocrine cells in culture throughthe use of specific growth factors (FIG. 1A). The first modificationreplaced Wnt3a with BIO-acetoxime (BIO). Wnt3a has been documented asbeing important for mesendoderm specification and BIO-acetoxime is aselective inhibitor of GSR-3β which indirectly acts as a Wnt3a agonistduring cell differentiation (33, 34). Second, nicotinamide was includedduring the last stage of differentiation because various publishedprotocols included this reagent due to strong evidence for its efficacyin pancreatic differentiation (35, 36).

To characterize the impact of the differentiation protocol on pancreaticendocrine specification in WT ES cells, cellular extracts were harvestedat various time points during the procedure (FIG. 1B). The mRNA levelsof various differentiation markers were assessed by RT-PCR for thedifferent experimental conditions. LIF withdrawal combined with theaddition of BIO and Activin A to the culture system caused a drasticdecrease in the levels of ES cell markers Nanog and Rex1 (FIG. 1B, lane6) compared to untreated and RA-treated ES cells (FIG. 1B, lanes 1 to4). Such a phenomenon was observed throughout the subsequent phases ofthe differentiation protocol (FIG. 1B, lanes 7 to 12). While robustexpression of glucagon, a functional marker of α-cells (37), wasobserved by day-14 (FIG. 1B, lane 8), somatostatin, a hormone secretedby δ-cells (37), was detectable as early as day-5 (FIG. 1B, lane 6).Insulin-1 (β-cell marker)(37) was detected by day-11 of thedifferentiation protocol but its expression fluctuated depending on theuses of HGF, IGF1, or both factors together during the endocrine celldifferentiation stage (FIG. 1B, lanes 9 to 11). The most consistentexpression of all 3 pancreatic endocrine differentiation markers testedwas observed by combining HGF and IGF1 with nicotinamide, from day-14 to17 (FIG. 1B, lane 12). Even though keeping ES cells in culture, atconfluence and in absence of LIF for 17 days, caused a decrease in Nanogand Rex1 expression, such conditions failed to induce any of thedifferentiation markers tested (FIG. 1B, lane 5).

These observations confirm the conversion of ES cells to endocrine cellsable to express pancreatic hormone-encoding genes, according to a methoddescribed previously (15). Such a biological model represents a powerfultool to investigate the role of RARβ at specific stages of pancreaticendocrine differentiation.

Example 3 RARβ Knockout delays Pdx1 Expression in Pancreatic EndocrineDifferentiation

As previously mentioned, the RA signaling, including the participationof RARβ, was suggested to be crucial for the onset of pancreaticendocrine differentiation (11, 20, 21, 24). In order to study thespecific role of RARβ in such a process, WT and RARβ KO mouse ES cellswere subjected to the endocrine differentiation protocol describedabove. RT-PCR analysis confirmed the absence of RARβ transcript in KOcells (FIG. 2A). The RARβ2 isoform, like Cyp26a1, represents aRA-inducible gene (38). This explains why stronger RARβ signal wasobserved in the presence of RA, in WT cells compared to untreated ones(FIG. 2A). RA-dependent Cyp26a1 expression was observed in both WT andRARβ KO ES cells, suggesting that KO cells are still responding to RAstimuli (FIG. 2A). Using this model of RARβ deletion, the inventorssought to determine the impact of such a retinoid receptor on theexpression of Pdx1, which consists in a master regulator of pancreaticcell fate (39-41).

WT and RARβ KO ES cells were differentiated into pancreatic endocrinecells, as described in FIG. 1, and indirect immunofluorescence stainingfor Pdx1 was performed at the different stages of the protocol (FIG.2B). Pdx1 expression was observed in WT differentiating cells by day-5,and was still present at all the other stages tested in a heterogeneouspattern (FIG. 2B). In contrast, Pdx1 protein was absent from nuclei ofdifferencing cells at day-5 and 11, and was only detected by day-14 ofthe protocol in RARβ null cells (FIG. 2B).

These observations suggest that the absence of RARβ in this cell culturesystem undergoing pancreatic differentiation engenders a delay in theinduction of Pdx1, which could potentially affect subsequent key stepsof endocrine specialization.

Example 4 Absence of RARβ Expression Impairs the Global PancreaticEndocrine Differentiation Process

Considering the finding that RARβ deletion in ES cells delays theexpression of Pdx1 during their specialization into pancreatic endocrinecells, the inventors decided to further investigate the impact of such aphenomenon on early, intermediate, and late molecular genetic eventsthroughout the differentiation process. As reported in many studies onreprogramming, decreased expression of pluripotency factors, includingNanog, in ES cells is essential for proper differentiation (42). Nanoglevels were previously shown to decrease around day-5 during thepancreatic endocrine differentiation protocol (FIG. 1B). A comparison ofNanog transcript levels in WT and RARβ KO differentiating cells, showeda sustained expression of this pluripotency factor in KO cells while itis severely repressed in WT controls (FIG. 3A). On the other hand, theexpression of Neurogenin-3 (Ngn3), a master transcription factor duringonset of pancreatic endocrine lineages (39, 41, 43), displayed a phasedinduction pattern in WT cells but was not induced in RARβ knockout (FIG.3A).

Like Ngn3, Paired-box 6 (Pax6) and Islet1 (Is1-1) represent twoimportant transcription factors in pancreatic islet celldifferentiation, which are expressed from intermediate (““mid””) toterminally differentiated (““late””) stages (39, 40, 44, 45). While nodifference were noted for Pax6 expression patterns, Is1-1 displayed adelayed expression peak in RARβ KO cells as compared to WT (day-14versus day-11) (FIG. 3B).

Finally, the expression of different functional endocrinedifferentiation markers such as, glucagon (Hcg; α-cells), insulin-1(Ins1; β-cells) and islet amyloid polypeptide (IAPP; β-cells) wasanalyzed in RARβ KO and WT differentiating cells (15, 46, 47) (FIG. 3C).In all cases, RARβ KO cells showed impaired expression of thosefunctional markers as compared to WT (FIG. 3C). Specifically, by day-17Gcg, Ins1, and Iapp respectively presented ˜5-fold (p=0.04), ˜120-fold(p=0.013), and ˜7-fold (p=0.0002) increases in WT differentiated cellsas compared to RARβ KO (FIG. 3C). Somatostatin (Sst), a functionalmarker of δ-cells (37) also displayed a decreased expression in RARβdeficient cells (not shown).

Taken together, these observations show that RARβ and retinoid signalingplay a central role in pancreatic endocrine differentiation byregulating the expression of certain master genes at early andintermediate stages of the specialization process, which as a resultimpairs the expression of functional markers of pancreatic islet cells.

Example 5 Deletion of RARβ Affects in Vivo Glucose Metabolism andPancreatic Islet Functionality

The tissue culture system used to study diverse steps of pancreaticendocrine differentiation provided important insights about the roleplayed by RARβ in such a physiological process. Specifically, theabsence of RARβ expression leads to decreased or delayed expression ofcrucial transcription factors involved in islet cell differentiation, aswell as decreased expression of functional differentiation markers(FIGS. 2 and 3). Thus, the inventors sought to validate the relevance ofthis finding in an in vivo model. A classical KO of both RARβ alleles inmice, generated and characterized by Ghyselinck et al. (26), was used tostudy the impact of such a deletion on pancreatic endocrine functions.By extracting pancreas from WT and RARβ-deficient mice, and performingindirect immunofluorescence staining for C-peptide, a by-product ofinsulin biosynthesis (48), and glucagon, the inventors observed adecrease (˜75%, p<0.0001) in the size of KO mice islets as compared toWT (FIG. 4A). Western blot analysis confirmed the decrease in C-peptideand glucagon expression in RARβ KO mice pancreas extracts as compared toWT controls (FIG. 4A). These observations demonstrate that RARβ KO micedisplay decreased pancreatic endocrine islet cell production and/ormaintenance, which could have major, deleterious effects on glucosemetabolism.

To assess the systemic effects of RARβ deletion on reduced insulin andglucagon-producing cells, mice of both groups were fasted for 15 hoursand blood glucose concentration was measured. While blood glucose levelsin WT were normal (between 70 and 105 mg/dL) (49), RARβ KO animals werefound to be in a hypoglycemic state, slightly below normal levels(61±4.7 mg/dL) (FIG. 4B) (50). In order to test the functionality ofβ-cells in both mice groups, a time-course blood glucose readingexperiment was performed which an intraperitonial injection of 2 mg/Kg(body weight) dextrose at time ““0””. Then, blood glucose clearance wasmonitored at 0, 15, 30, 45, 60, and 120 minutes in WT and RARβ KO mice.We observed that blood glucose was metabolized faster in WT mice, ascompared to KO (FIG. 4B). Moreover, the average blood glucose levels inRARβ KO mice 120 min after the dextrose injection was significantlyhigher (˜30%, p=0.014) than in WT group, suggesting a lower glucosetolerance in animals lacking such a retinoid receptor (FIG. 4B).

As described in the Examples, by using an ES cell-based directeddifferentiation system (Examples 2-4) and an in vivo gene knockout model(Example 5), the inventors demonstrated the crucial role for RARβ inproper pancreatic endocrine cell differentiation. In both cases, theabsence of RARβ led to a decrease in terminal differentiation andfunctional markers, such as insulin and glucagon production. In mice,RARβ deletion resulted in impaired glucose metabolism, characterized byhypoglycemia and glucose intolerance. Taken together, these findingsindicate that reduced RARβ and retinoic acid signaling are key factorsin glucose metabolism disorders, such as diabetes mellitus type I andII. Hence, the administration of agonists of the RARβ receptor canprevent or treat such disorders.

The study described in Example 2 leads to the conclusion that Pdx1expression, during the pancreatic differentiation process, was delayedin the absence of RARβ (FIG. 2). Such a transcription factor representsa key player in the early determination of pancreatic progenitors andbud expension (39, 40, 51, 52). A previous study reported that RAdirectly induces Pdx1 expression in ES cells (51). Strengthening such astatement, ChIP-chip analyses performed on F9 teratocarcinoma cellsrevealed the presence of a putative retinoic acid response element(RARE) located at ˜3 kb upstream of the transcription start site of Pdx1(not shown). That Pdx1 expression is delayed but not fully suppressed inRARβ-null ES cells opens a door on possible compensatory mechanismsexerted by other RARs. It has been previously noted that RARβ transcriptlevels are increased at stages of endocrine differentiation, while apeak of RARα expression is associated with late differentiation stages(24). Possibly RARα and β together participate in the Pdx1 biphasicexpression pattern, as reviewed by Soria (39). Thus, suppressing RARβwould result exclusively in a late Pdx1 expression as observed intreated RARβ KO cells (FIG. 2).

Pdx1 mis-expression was previously associated with severe β-celldysfunction and increased cell death (53). Accordingly, RARβ KO caused areduction in β-cell terminal differentiation markers' expression, suchas Ins1 and Iapp in the cell culture system (FIG. 3), as well as adecreased number of C-peptide expressing cells in RARβ null-micepancreatic islets (FIG. 4). Recent findings by Dalgin et al. (54) alsolinked RA signaling and endocrine cell fate. Although the authorsclaimed that β-cell progenitors differentiate as α-cells in RAdownstream target mnx1 morphants, the data reported here suggest thatRARβ KO induces a decrease in α-cell differentiation, characterized byreduced expression of glucagon in the cell culture system (FIG. 3) andRARβ null mice (FIG. 4). Thus, the effect observed on islet cells in theabsence of RARβ could be attributed to the role of RA signaling in earlypancreatic differentiation events rather than lineage-specific terminaldifferentiation.

Like Pdx1, the bHLH transcription factor Neurogenin3 (Ngn3) constitutesanother key player in the commitment of endoderm to pancreaticprecursors (40, 43, 47). Among the cascade of transcription factorsinvolved in pancreas development, Ngn3 is the earliest to be expressedin the endocrine differentiation pathway (40, 55). While no linksbetween RA signaling and Ngn3 expression was reported in the literature,RARβ KO cells displayed decreased levels of this transcription factorduring pancreatic differentiation (FIG. 3). Thus, the impact of RARβdeletion on Ngn3 could be indirect and involving the participation ofintermediate factors.

Pax6 and Is1-1 represent two major transcription factors having a rolein endocrine lineage specification after bud formation (45, 56)Considering that Pax6 expression is not affected by RARβ KO, and thatthe Is1-1 peak of expression is only delayed by such a deletion, itappears that absence of RA signaling through RARβ is insufficient tocompletely abrogate endocrine differentiation, but may lead tosignificant defects in islet cell function.

The observations reported here indicate that the absence of RARβexpression impairs development and maintenance of pancreatic islets invivo (FIG. 4). In mammals, glucose intolerance is characterized bysustained high blood glucose levels (above 140 mg/dL) during at leasttwo hours, while hypoglycemia is decreed when blood concentration goesbelow 70 mg/dL (50, 57). Blood glucose assessment: 1) after 15 h fastingand 2) upon dextrose injection led us to suggest that RARβ-null micehave a predisposition to fasting hypoglycemia and increased glucoseintolerance, two conditions associated with diabetes mellitus (58).

Close correlations have been made between dietary habits and diabetes,especially for type II (59). Considering the role of RARβ in pancreaticendocrine cell differentiation, and that the RARβ gene itself isup-regulated by retinoic acid, a sustained vitamin A deficient dietcould lead to insufficient islet cell turnover, and eventually todiabetes. RARβ expression is also known to depend on epigeneticregulation (60, 61). For instance, aberrant hypermethylation of variouspromoter elements was reported in different pancreatic disorders such ascancer, diabetes, and chronic pancreatitis (62-64). Therefore,epigenetic silencing of RARβ or other associated effectors could play arole in the onset of certain cases of diabetes.

The production of insulin secreting endocrine cells from ES cells usingRA-based protocols is proposed as a promising tool for diabetic therapy(9). However, ensuring accurate vitamin A consumption and proper RAsignaling via RARβ represent new avenues to prevent or treat diabeticdisorders. In particular, the administration of an RARβ agonist would bea specifically targeted method of enhancing this RARβ signaling toprevent or treat diabetic disorders. Taken together, these findings shedlight on the role of RARβ in pancreatic endocrine differentiation, whichconsequently affects in vivo blood glucose metabolism.

Example 6 RARβ Agonist Treatment Preparation

Preparation of AC261066 (a RARβ agonist from Tocris) solution. AC261066was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 1.5mg/ml, and diluted in the drinking water for mice to the finalconcentration of 1.5 mg/100 ml.

Mice, diet, and drug treatment. WT male C57/BL6 male mice weremaintained on either a standard laboratory chow-fed diet (CFD) with 13%kcal fat, (diet#5053, Lab Diet, Inc, St. Louis, Mo.) or a high fat,western style diet (HFD) with 60% kcals from fat, (diet #58126, LabDiet. Inc., St. Louis, Mo.) for 4 months. One month after the start ofthe high fat diet treatment, the high fat diet group was further splitinto 2 groups for 3 months: i) high fat diet and the drinking watercontaining 1% DMSO; ii) high fat diet and the drinking water containing1.5 mg/100 ml AC261066, a specific RARβ agonist. Then mice weresacrificed by cervical dislocation. Blood and various tissue sampleswere harvested.

Example 7 Pancreas

Semi-Quantitate PCR. Total RNA was extracted from mouse tissues usingTRIzol reagent (Life technologies) and (1 μg) was used to synthesizecDNA. cDNA synthesis was performed at 42° C. for 1 h in a final volumeof 20 μl using qScript (Quanta, Md.). Semi-quantitative PCR wereperformed Taq DNA polymerase (Invitrogen, Calif.). Three step PCR was nmas follows: 94° C. for 30 s, 58-64° C. for 45 s for primer annealing and72° C. for 1 min for primer extension. The number of cycles for eachprimer pair for amplification in the linear range was determinedexperimentally. PCR products were resolved on 2% agarose gels andvisualized by staining with ehtidium bromide. Primers for geneexpression used were as follows: RARβ2, F: 5′-TGGCATTGTTTGCACGCTGA-3′(SEQ 1D No. 25), R: 5′-CCCCCCTTTGGCAAAGAATAGA-3′ (SEQ ID No. 26),CYP26A1, F: 5′-CTTTATAAGGCCGCCCAGGTTAC-3′ (SEQ ID No. 27), R:5′-CCCGATCCGCAATTAAAGATGA-3′ (SEQ ID No. 28), LRAT, F:5′-TCTGGCATCTCTCCTACGCTG-3′ (SEQ ID No. 29), R:5′-GTTCCAAGTCCTTCAGTCTCTTGC-3′ (SEQ ID No. 30), INS2, F:5′-TGTGGGGAGCGTGGCTTCTTCT-3′ (SEQ ID No. 31), R:5′-CAGCTCCAGTTGTGCCACTTGT-3′ (SEQ ID No. 32), HPRT,F:5′-TGCTCGAGTGTGATGAAGG-3′ (SEQ ID No. 33), R:5′-TCCCTGTTGACTGGTCATT-3′(SEQ ID No. 34).

Analysis of pancreatic retinoids. The frozen pancreas tissue samples(˜100 mg) were homogenized in 500 μl cold phosphate-buffered saline(PBS). In addition, 100 μl serum was diluted in cold PBS to total volumeof 500 μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millennium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

4-hydroxynonerial (4-HNE) staining. Paraffin-embedded sections (from twoto four mice per group) were deparaffinized and rehydrated, and antigenretrieval was performed using an antigen unmasking solution (VectorLaboratories, H-3300). After quenching endogenous peroxidase with 3%H₂O₂, the tissue sections were blocked with the blocking reagent (fromthe M.O.M. kit from Vector Laboratories). Then, tissue sections wereincubated with a 4-HNE antibody (1:400; mouse monoclonal antibody;Abeam, ab48506) overnight at 4° C. The sections were then incubated withsecondary antibodies (1:200, anti-mouse IgG from the M.O.M kit). As anegative control, sections were stained without incubation with primaryantibodies. The signals were visualized based on a peroxidase detectionmechanism with 3,3-diaminobenzidine (DAB) used as the substrate.

Retinoid levels in pancreatic tissue. Our HPLC analysis revealed thatthat pancreata from HF-fed obese mice had dramatically decreased levelsretinol (VA, vitamin A) compared to CF (control diet) controls (FIG. 5).Retinyl palmitate was undetectable in pancreata tissue from HF-fed mice(FIG. 5), showing profound pancreas vitamin A deficiency.

Serum retinol from mice on a high fat diet vs. control diet compared tothe pancreas retinol and retinyl palmitate levels from mice on a highfat vs. control diet. The serum retinol levels are similar or a hithigher in the HF diet mice, but the pancreas retinol levels are muchlower in the HF diet mice, showing vitamin A deficiency in the pancreaseven in the presence of normal serum vitamin A (FIG. 6).

AC261066 decreases oxidative stress levels in the pancreas from HF-fedmice. High fat diet results in excessive reactive oxygen species (ROS)production that triggers inflammatory responses and subsequent injuriesin many tissues. Therefore, we examined the levels of 4-hydroxynonenal(4-HNE), an α,β-unsaturated hydroxyalkenal that is produced by lipidperoxidation in cells during oxidative stress, and is a marker ofoxidative stress caused by reactive oxygen species (ROS) in thepancreas. The pancreatic islets from HF-fed mice showed an increase inthe 4-HNE levels compared to the chow-fed controls (FIG. 7). Thepancreatic islet samples from the high fat diet plus AC261.066 groupexhibited markedly lower 4-HNE staining intensity levels compared toHF-vehicle treated mice (FIG. 7).

AC261066 does diminish pancreatic islet insulin expression. Next weexamined the changes to pancreatic expression of endocrine hormones inCF, HF and HF+AC261066 fed mice. Pancreatic islets stained forpro-insulin c-peptide (green) and glucagon (red) revealed that isletsfrom HF and HF+AC261066 fed mice showed a marked increase in c-peptidestaining compared to control diet controls (FIG. 8). AC261066 slightlydecreased c-peptide level in the HF diet mice.

AC2621066 increased pancreatic mRNA expression of RARβ in obese andvitamin A deficient mice. Consistent with our HPLC data demonstratingthat pancreata tissue from HF-fed, obese nice had significantlydecreased VA (vitamin A) levels, and significantly decreased mRNA levelsof the VA responsive gene and VA signaling transcription factor, RARβ.RAR β was decreased in pancreata of HF-fed obese mice compared tocontrol diet fed mice (FIG. 9). mRNA levels of RARβ in pancreataHF-AC261066 treated mice were increased compared to HF-vehicle treatedmice (FIG. 9), and near levels observed in non-obese controls,suggesting that AC261066 can prevent or reverse the loss of VA signalingin VA depleted tissue. Similar findings in vitamin A deficient mice,FIG. 10.

Example 8 Liver

Hematoxylin and Eosin Staining. At sacrifice, fresh mouse liver sampleswere fixed in 4% formaldehyde solution for 24 hr and embedded inparaffin blocks. Liver paraffin sections were cut 5 microns thick andmounted on glass slides and stained with hematoxylin and eosin (H and E)using standard protocols.

Combined oil red O and Immunofluorescence. Staining. Fresh mouse liversamples were embedded in optimal cutting temperature (OCT) medium andimmediately frozen to −70 centigrade. Cryosections were then fixed in 4%formaldehyde for 1 hr at room temp. Slides were then rinsed three timesin deionized water (dH2O) for 30 s, followed by treatment with 0.5%Triton X-100 in PBS for 5 min. Sections were then washed three timeswith PBS for 5 min. Samples were with incubated 2% bovine serum albumin(BSA) for 30 min at room temperature to block for unspecific antibodybinding. Following blocking, sections were washed three times in PBS andincubated with mouse monoclonal antibody against α-SMA (1:500) (Dako,Inc) for 24 h at 4° C. After 24 h sections were washed three times in PBand incubated with Alexa-Flour-488 anti-mouse secondary anti-body(1:500) (Invitrogen, Inc) for 30 min at room temperature. Sections werethen washed three times in PBS and incubated with working strengthoil-red O solution for 30 minutes at room temperature. Sections werethen rinsed for 30 minutes under running tap water and cover-slippedwith Vectashield hard mount plus DAPI (Vector Labs, Inc).

Semi-Quantitate PCR (Liver). Total RNA was extracted from mouse tissuesusing TRIzol reagent (Lite technologies) and (1μg) was used tosynthesize cDNA. cDNA synthesis was performed at 42° C. for 1 h in afinal volume of 20 μl using qScript (Quanta, Md.). Semi-quantitative PCRwere performed Tag DNA polymerase (Invitrogen, Calif.). Three step PCRwas run as follows: 94° C. for 30 s, 58-64° C. for 45 s for primerannealing and 72° C. for 1 min for primer extension. The number ofcycles for each primer pair for amplification in the linear range wasdetermined experimentally. PCR products were resolved on 2% agarose gelsand visualized by staining with ehtidium bromide. Printers for geneexpression used were as follows: RARβ2, F: 5′-TGGCATTGTTTGCACGCTGA-3′(SEQ ID No. 25), R: 5′-CCCCCCTTTGGCAAAGAATAGA-3′ (SEQ ID No. 26),CYP26A1, F: 5′-CTTTATAAGGCCCGCCCAGGTTAC-3′ (SEQ ID No. 27), R:5′-CCCGATCCGCAATTAAAGATGA-3′ (SEQ ID No. 28), LRAT, F:5′-TCTGGCATCTCTCCTACGCTG-3′ (SEQ ID No, 29), R:5′-GTTCCAAGTCCTTCAGTCTCTTGC-3′ (SEQ ID No. 30), INS2, F:5′-TGTGGGGAGCGTGGCTTCTCT-3′ (SEQ ID No. 31), R:5′-CAGCTCCAGTTGTGCCACTTGT-3′ (SEQ ID No. 32). TNFα, F:5′-CCTGTAGCCCACGTCGTAG-3′ (SEQ ID No. 35), R:5′-GGGAGTAGACAAGGTACAACCC-3′ (SEQ ID No. 36), MCP1, F:5′-TTAAAAACCTGGATCGGAACCAA-3′ (SEQ ID No. 37), R:5′-GCATTAGCTTCAGATTTACGGGT-3′ (SEQ ID No. 38), HPRT,F:5′-TGCTCGAGTGTGATGAAGG-3′ (SEQ IF) No. 33),R:5′-TCCCTGTTGACTGGTCATT-3′ (SEQ ID No. 34).

Serum triglyceride level measurement. The analysis of serum triglyceridelevels was carried out using a bichromatic assay at the Laboratory ofComparative Pathology of the Memorial Sloan-Kettering Cancer Center.Chow-fed diet (CFD) n=2; high fat diet (HFD) n=3; high fat diet+AC261066(HFDAC) n=5.

Analysis of serum and liver retinoids. The frozen liver tissue samples(˜100 mg) were homogenized in 500 μl cold phosphate-buffered saline(PBS). In addition, 100 μl serum was diluted in cold PBS to total volumeof 500 μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millennium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

4-hydroxynonenal (4-HNE) staining. Paraffin-embedded sections (from twoto four mice per group) were deparaffinized and rehydrated, and antigenretrieval was performed using an antigen unmasking solution (VectorLaboratories, H-3300). After quenching endogenous peroxidase with 3%H2O2, the tissue sections were blocked with the blocking reagent (fromthe M.O.M. kit from Vector Laboratories). Then, tissue sections wereincubated with a 4-HNE antibody (1:400; mouse monoclonal antibody;Abeam, ab48506) overnight at 4° C. The sections were then incubated withsecondary antibodies (1:200, anti-mouse IgG from the M.O.M kit). As anegative control, sections were stained without incubation with primaryantibodies. The signals were visualized based on a peroxidase detectionmechanism with 3,3-diaminobenzidine (DAB) used as the substrate.

Analysis of serum and liver retinoids. The frozen liver tissue samples(˜100 mg) were homogenized in 500 μl cold phosphate-buffered saline(PBS). In addition, 100 μl serum was diluted in cold PBS to total volumeof 500 μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millenium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

AC261066 diminished hepatic steatosis. H and E staining of liversections from treatment mice revealed that 4 months of a HF westernstyle diet lead to increased hepatocyte lipid accumulation in HF-fedmice compared to CFD-fed mice (FIG. 11). HF-fed mice treated withAC261066 showed marked decreased hepatocyte lipid infiltration comparedto HF-vehicle treated mice (FIG. 11). HF-fed mice treated with a RAR γligand (CD1530) showed no decrease in hepatic lipid accumulation (FIG.11).

AC261066 diminishes hepatic gene expression of alpha-SMA (alpha-smoothmuscle actin) and SREBP1c. Consistent with our immunofluorescencemicroscopy showing that α-SMA protein is decreased in HF-AC261011 fedmice compared to HF-vehicle controls, hepatic mRNA levels of alpha-SMAwere also decreased in livers of HF-AC261011 fed mice, but not in thelivers of HF-CD1530 treated mice (FIG. 12). We also measured mRNAexpression of SREBP1-c, which codes for a transcription factorresponsible for de novo synthesis of triglyceride and is oftenover-expressed in livers of animals with experimentally induced NAFLD.Our analysis revealed that mRNA levels of SREBP1-c are markedly higherin livers of HF-fed and HF-fed CD1530 treated mice, but not in livers ofHF-AC261011 treated mice (FIG. 12).

AC261066 diminishes hepatic stellate cell (HSC) activation. Liversections co-stained with the neutral lipid stain oil-red-o were inagreement with the H and E staining, demonstrating that HF-fed obesemice had ectopic accumulation of hepatic lipids (red) compared to CFcontrols (FIG. 13). Livers of HF-AC261066-fed mice had marked diminishedhepatic lipid accumulation compared to HF vehicle-fed mice (FIG. 13).This effect was not observed in the livers of HF-fed mice treated withthe CD1530 (RARγ agonist).

Activated HSCs contribute to normal liver tissue repair processes, butunresolved. HSC activation can lead to fibrotic lesion formation and theprogression of steatosis to advanced NAFLD, such as non-alcoholicsteatohepatitis (NASH). To examine whether HF-fed obese mice exhibitedevidence of increased activation of HSCs we stained liver sections withan α-SMA antibody. This analysis revealed the livers of HF-fed mice hadincreased α-SMA positive (green) staining compared to lean, CF controls.α-SMA positive areas tended to cluster in areas with hepatocyte lipidinfiltration (FIG. 13). Compared to HF-fed mice, livers ofHF-fed-AC261066 treated mice had decreased intensity and regions ofα-SMA positive staining (FIG. 13). Moreover, clustering α-SMA in lipidpositive (red) regions was not observed in liver of HF-AC261066 treatedmice. Livers of HF-fed CD1530 treated mice had no evidence decreasedlipid accumulation or α-SMA expression intensity or patterns compared toHF fed-vehicle treated mice.

AC261066 diminishes hepatic gene expression of pro-inflammatorymediators. NAFLD is typically associated with increased hepaticexpression of pro-inflammatory cytokines and mediators such as themonocyte chemokine MCP-1 and the cytokine TNF-α. We examined expressionof these genes in livers of CF and HF-fed mice. Our analysis revealedthat mRNA levels of both MCP-1 and TNF-α were markedly elevated inlivers of HF-fed mice HF-fed CD1530 treated mice, but not in livers ofHF-fed AC261066 treated mice (FIG. 14).

AC261066 does not elevate serum triglyceride levels. We examined thetriglyceride levels in mouse serum samples because elevatedtriglycerides are a risk factor for cardiovascular disease. As shown inFIG. 15, HF or HF+AC261066 feeding does not affect serum triglyceridelevels compared CF controls. This suggests that AC261066 does notincrease risk for cardiovascular disease and suggests that the liverlipid lowering effect of AC261066 does not correlate with increasedhepatic lipid export.

AC261066 partially reverses depletion of VA in livers of HF-fed ObeseMice. The liver stores approximately 80-90% of total body VA, thereforewe conducted HPLC to determine the tissue levels of the major storageform of VA, retinyl-palmitate and of all-trans retinol in lean CF, HFand HF+AC261066 fed mice. Our analysis revealed that levels ofretinyl-palmitate and retinol were decreased by 97% and 92% in livers inHF-fed, obese mice compared to lean, CF controls (FIG. 16). Serum levelsof the major circulating form of VA, all-trans retinol were notdifferent between CF, HF and HF+AC261066 fed mice, suggesting thatHF-driven obesity leads to tissue VA depletion which is not reflected byserum VA levels.

Livers of mice fed HF+AC261066 and CD1530 also had significantly loweredretinyl palmitate and retinol compared to controls, however compared toHF-vehicle treated mice, we observed 55% higher levels of retinylpalmitate in the livers from HF-AC261066 fed mice, while retinylpalmitate levels in the liver of HF′CD1530 treated mice were almost 48%lower than livers from HF-vehicle treated mice (FIG. 16). This suggeststhat longer administration of AC261066 to HF-fed obese mice may havesignificantly reversed HF-obesity driven liver VA depletion.

Oxidative stress level, as assessed by 4-hydroxynoneal (4-HNE), is lowerin the liver from the high fat diet plus AC261066 group than that in thehigh fat diet group. High fat diet results in excessive reactive oxygenspecies (ROS) production that triggers inflammatory responses andsubsequent injuries in many tissues. Therefore, we examined the levelsof 4-hydroxynonenal (4-HNE), an α,β-unsaturated hydroxyalkenal that isproduced by lipid peroxidation in cells during oxidative stress, and isa marker of oxidative stress caused by reactive oxygen species (ROS) inthe liver. The liver from the high fat diet group showed a largeincrease in the 4-HNE levels compared to the control fat diet group, andthe liver samples from the high fat diet plus AC261066 group exhibitedlower 4-HNE levels than those from the high fat diet group (FIG. 17).

Example 9 Kidney

Hematoxylin and Eosin Staining. At sacrifice, fresh mouse liver sampleswere fixed in 4% formaldehyde solution for 24 hr and embedded inparaffin blocks. Kidney paraffin sections were cut 5 microns thick andmounted on glass slides and stained with hematoxylin and eosin (H and E)using standard protocols.

Combined oil red O and Immunofluorescence staining. Fresh mouse kidneysamples were embedded in optimal cutting temperature (OCT) medium andimmediately frozen to −70 centigrade. Cryosections were then fixed in 4%formaldehyde for 1 hr at room temp. Slides were then rinsed three timesin deionized water (dH2O) for 30 s, followed by treatment with 0.5%Triton X-100 in PBS for 5 min. Sections were then washed three timeswith PBS for 5 min. Samples were with incubated 2% bovine serum albumin(BSA) for 30 min at room temperature to block for unspecific antibodybinding. Following blocking, sections were washed three times in PBS andincubated with mouse monoclonal antibody against α-SMA (1:500) (Dako,Inc) for 24 h at 4° C. After 24 h sections were washed three times in PBand incubated with Alexa-Flour-488 anti-mouse secondary anti-body(1:500) (Invitrogen, Inc) for 30 min at room temperature. Sections werethen washed three times in PBS and incubated with working strengthoil-red O solution for 30 minutes at room temperature. Sections werethen rinsed for 30 minutes under finning tap water and cover-slippedwith Vectashield hard mount plus DAPI (Vector Labs, Inc).

Semi-Quantitative PCR. Total RNA was extracted from mouse tissues usingTRIzol reagent (Life technologies) and (1 μg) was used to synthesizecDNA. cDNA synthesis was performed at 42° C. for 1 h in a final volumeof 20 μl using qScript (Quanta, Md.). Semi-quantitative PCR wereperformed Tag DNA polymerase (Invitrogen, Calif.). Three step PCR wasrun as follows: 94° C. for 30 s, 58-64° C. for 45 s for primer annealingand 72° C. for 1 min for primer extension. The number of cycles for eachprimer pair for amplification in the linear range was determinedexperimentally, PCR products were resolved on 2% agarose gels andvisualized by staining with ehtidium bromide. Primers for geneexpression used were as follows: RARβ2, F: 5′-TGGCATTGTTTGCACGCTGA-3′(SEQ ID No. 25), R: 5′-CCCCCCTTTGGCAAAGAATAGA-3′ (SEQ ID No. 26),CYP26A1, F: 5′-CTTTATAAGGCCGCCCAGGTTAC-3′ (SEQ ID No. 27), R:5′-CCCGATCCGCAATTAAAGATGA-3′ (SEQ ID No. 28). TNFα, F:5′-CCTGTAGCCCACGTCGTAG-3′ (SEQ ID No. 35), R:5′-GGGAGTAGACAAGGTACAACCC-3′ (SEQ ID No. 36), HPRT, F:5′-TGCTCGAGTGTGATGAAGG-3′ (SEQ ID No. 33), R:5′-TCCCTGTTGACTGGTCATT-3′(SEQ ID No. 34).

Analysis of kidney retinoids. The frozen kidney tissue samples (˜100 mg)were homogenized in 500 μl cold phosphate-buffered saline (PBS). Inaddition, 100 μl serum was diluted in cold PBS to total volume of 500μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millennium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

4-hydroxynonenal (4-HNE) staining. Paraffin-embedded sections (from twoto four mice per group) were deparaffinized and rehydrated, and antigenretrieval was performed using an antigen unmasking solution (VectorLaboratories, H-3300). After quenching endogenous peroxidase with 3%H₂O₂, the tissue sections were blocked with the blocking reagent (fromthe M.O.M. kit from Vector Laboratories). Then, tissue sections wereincubated with a 4-HNE antibody (1:400; mouse monoclonal antibody;Abeam, ab48506) overnight at 4° C. The sections were then incubated withsecondary antibodies (1:200, anti-mouse IgG from the M.O.M kit). As anegative control, sections were stained without incubation with primaryantibodies. The signals were visualized based on a peroxidase detectionmechanism with 3,3-diaminobenzidine (DAB) used as the substrate.

AC261066 diminished renal lipid accumulation. H and E staining of kidneysections from treatment mice revealed that 4 months of a HF westernstyle diet lead to increased renal lipid accumulation in HF-fed micecompared to CFD-fed mice (FIG. 18). HF-fed mice treated with AC261066showed markedly decreased renal lipid infiltration compared toHF-vehicle treated mice (FIG. 18). HF-fed mice treated with a RAR γligand (CD1530) showed no decrease in renal lipid accumulation (FIG.18).

AC261066 diminishes renal expression of alpha-SMA. Kidney sectionsco-stained with the neutral lipid stain oil-red-o were in agreement withthe H and E staining, demonstrating that HF-fed obese mice had ectopicaccumulation of renal lipids (red) compared to CF controls (FIG. 18).Kidneys of HF-AC261066-fed mice had marked diminished hepatic lipidaccumulation compared to HF vehicle-fed mice (FIG. 19). Alpha-SMA isrequired for normal kidney tissue repair processes, but uncheckedalpha-SMA secretion can lead to fibrotic lesion formation and theprogression of advanced renal disease. As expected kidney sectionsstained with the neutral lipid stain oil-red-o (red) showed markedincrease in renal lipid droplets in kidneys of HF-fed mice compared tocontrol fed mice. In agreement with our H and F histological analysis,kidney sections from HF+AC261066 treated mice had comparably less oilred o positive areas, α-SMA (green) staining also revealed that kidneysof HF-fed mice had increased α-SMA positive areas compared to controlfed mice (FIG. 19). This increase in α-SMA positive areas was notobserved in kidneys of HF+AC261066 treated mice.

Retinoid levels in kidneys. Our HPLC analysis of kidney tissuedemonstrated that HF-fed obese mice had significantly decreased levelsof kidney retinyl palmitate and retinol compared to chow fed controls(FIG. 20).

AC261066 diminishes kidney gene expression of pro-inflammatorymediators. Fibrosis is associated increased renal expression ofpro-inflammatory cytokines and mediators. We examined whether kidneys ofHF-fed mice had evidence of inflammation marked by increased expressionof inflammatory cytokines such as TNF-α. Our analysis revealed that mRNAlevels of TNF-α were markedly elevated in livers of HF-fed mice, but notin livers of HF-fed AC261066 treated mice (FIG. 21).

AC261066 increased kidney gene expression of RARβ2. Consistent with theHPLC data demonstrating that VA levels are diminished in kidney ofHF-fed mice, our kidney gene expression analysis revealed that RARβ2mRNA is markedly decreased in the kidney of HF-fed mice (FIG. 21).Kidney's from HF-AC261066 did not have decreased RARβ2 mRNA levels (FIG.21).

Oxidative stress level, as assessed by 4-hydroxyrioneal (4-HNE), islower in the kidneys from the high fat diet plus AC261066 group thanthat in the high fat diet group. High fat diet results in excessivereactive oxygen species (ROS) production that triggers inflammatoryresponses and subsequent injuries in many tissues. Therefore, weexamined the levels of 4-hydroxynonenal (4-HNE), an α,β-unsaturatedhydroxyalkenal that is produced by lipid peroxidation in cells duringoxidative stress, and is a marker of oxidative stress caused by reactiveoxygen species (ROS) in the kidneys The kidneys from the high fat dietgroup showed a large increase in the 4-HNE levels compared to thecontrol fat diet group, and the kidneys from the high fat diet plusAC261066 group exhibited lower 4-HNE levels than those from the high fatdiet group (FIG. 22)

Example 10 Testes

Semi-Quantitative PCR. Total RNA was extracted from mouse tissues usingTRIzol reagent (Life technologies) and (1 μg) was used to synthesizecDNA. cDNA synthesis was performed at 42° C. for 1 h in a final volumeof 20 μl using ((Script (Quanta, Md.). Semi-quantitative PCR wereperformed Taq DNA polymerase (Invitrogen, Calif.). Three step PCR wasmin as follows: 94° C. for 30 s, 58-64° C. for 45 s for primer annealingand 72° C. for 1 min for primer extension. The number of cycles for eachprimer pair for amplification in the linear range was determinedexperimentally. PCR products were resolved on 2% agarose gels andvisualized by staining with ehtidium bromide. Primers for geneexpression used were as follows: RARβ2, F: 5′-TGGCATTGTTTGCACGCTGA-3′(SEQ ID No. 25), R: 5′-CCCCCCTTTGGCAAAGAATAGA-3′ (SEQ ID No. 26).CYP26A1, F: 5′-CTTTATAAGGCCGCCCAGGTTAC-3′ (SEQ ID No. 27), R:5′-CCCGATCCGCAATTAAAGATGA-3′ (SEQ ID No. 28), APRT, F:5′-TGCTCGAGTGTGATGAAGG-3′ (SEQ ID No. 33), R:5′-TCCCTGTTGACTGGTCATT-3′(SEQ ID No. 34).

Analysis of testes retinoids. The frozen kidney tissue samples (˜100 mg)were homogenized in 500 μl cold phosphate-buffered saline (PBS). Inaddition, 100 μl serum was diluted in cold PBS to total volume of 500μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millennium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

Retinoid levels in testes. Our HPLC analysis of testes demonstrated thatHF-fed obese mice had significantly decreased levels of retinylpalmitate (storage form of VA) and decreased retinol compared to chowfed controls (FIG. 23).

Testes of HF-fed Mice have decreased expression of VA relevant genesexpression. Consistent with the HPLC data demonstrating that VA levelsare diminished in kidney of HF-fed mice, our testes gene expressionanalysis revealed that RARβ2 and CYP26A1, and RAR gamma2 mRNAs aremarkedly decreased in the testes of 14F-fed mice (FIG. 24).

RARβ agonist AC55649 is prepared in the same way and is used to treatmice as described in Examples 6-10.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices and materials are herein described. All publications mentionedherein are hereby incorporated by reference in their entirety for thepurpose of describing and disclosing the materials and methodologiesthat are reported in the publication which might be used in connectionwith the invention.

REFERENCES

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What is claimed is:
 1. A method of treating or preventing a pancreaticdisease in a subject comprising administering to said subject vitamin Aor an agonist of retinoic acid receptor-beta (RARβ).
 2. The method ofclaim 1, wherein said pancreatic disease is associated with obesity. 3.The method of claim 1, wherein said pancreatic disease is caused by ahigh fat diet.
 4. The method of claim 1, wherein said pancreatic diseaseis diabetes.
 5. The method of claim 1, wherein said diabetes is type Ior type II diabetes, or gestational diabetes.
 6. The method of claim 1,wherein said pancreatic disease is associated with reduced vitamin Alevel in the pancreas.
 7. A method of treating or preventing thedegeneration of pancreatic beta cells in a subject comprisingadministering to said subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).
 8. A method of maintaining or improving thefunction of pancreatic beta cells in a subject comprising administeringto said subject vitamin A or an agonist of retinoic acid receptor-beta(RARβ).
 9. A method of maintaining or improving pancreatic insulinsecretion in a subject comprising administering to said subject vitaminA or an agonist of retinoic acid receptor-beta (RARβ).
 10. A method ofmaintaining or improving insulin sensitivity in a subject comprisingadministering to said subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).
 11. A method of maintaining or improving the levelof glucagon in a subject comprising administering to said subjectvitamin A or an agonist of retinoic acid receptor-beta (RARβ).
 12. Amethod of treating or preventing fat deposit in a subject comprisingadministering to said subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).
 13. A method of treating or preventinginflammation in a subject comprising administering to said subjectvitamin A or an agonist of retinoic acid receptor-beta (RARβ).
 14. Amethod of decreasing oxidative stress in a subject comprisingadministering to said subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).
 15. The method of claim 12, 13 or 14, wherein saidfat deposit, inflammation or oxidative stress is in an organ of saidsubject.
 16. The method of claim 15, wherein said organ is pancreas,liver, kidney or testes.
 17. A method of increasing RARβ level in asubject comprising administering to said subject vitamin A or an agonistof retinoic acid receptor-beta (RARβ).
 18. The method of claim 17,wherein said RARβ level is increased in an organ.
 19. The method ofclaim 18, wherein said organ is pancreas, liver, kidney or testes.
 20. Amethod of decreasing the level of an inflammatory mediator in a subjectcomprising administering to said subject vitamin A or an agonist ofretinoic acid receptor-beta (RARβ).
 21. The method of claim 20, whereinsaid production of said inflammatory mediator is decreased.
 22. Themethod of claim 20, wherein said secretion of said inflammatory mediatoris decreased.
 23. The method of claim 20, wherein said inflammatorymediator is monocyte chemotactic protein (mcp-1) or tumor necrosisfactor alpha (tnf-α).
 24. The method of anyone of claims 20-23, whereinsaid level of inflammatory mediator is decreased in an organ.
 25. Themethod of claim 24, wherein said organ is pancreas, liver, kidney ortestes.
 26. A method of treating or preventing a liver disease in asubject comprising administering to said subject vitamin A or an agonistof retinoic acid receptor-beta (RARβ).
 27. The method of claim 26,wherein said liver disease is associated with obesity.
 28. The method ofclaim 26, wherein said liver disease is associated with a high fat diet.29. The method of claim 26, wherein said liver disease is fatty liverdisease (FLD), liver fibrosis, or hepatic steatosis.
 30. The method ofclaim 26, wherein said liver disease is associated with reduced vitaminA level in the liver.
 31. The method of claim 26, wherein said liverdisease is non-alcoholic FLD (NAFLD), alcohol associated FLD, ornon-alcoholic steatohepatitis (NASH).
 32. A method of decreasing theactivation of hepatic stellate cells (HSCs) in a subject comprisingadministering to said subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).
 33. A method of decreasing the level of hepaticreactive oxygen species (ROS) in a subject comprising administering tosaid subject vitamin A or an agonist of retinoic acid receptor-beta(RARβ).
 34. A method of decreasing the level of alpha smooth muscleactin (α-SMA) in a subject comprising administering to said subjectvitamin A or an agonist of retinoic acid receptor-beta (RARβ).
 35. Themethod of claim 34, wherein said subject has a disease of non-alcoholicFLD (NAFLD), alcohol associated FLD, or non-alcoholic steatohepatitis(NASH).
 36. A method of increasing the level of lethicin:retinolacyltransferase (LRAT) in the liver of a subject comprisingadministering to said subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).
 37. A method of increasing the level of RARβ inthe liver of a subject comprising administering to the subject vitamin Aor an agonist of retinoic acid receptor-beta (RARβ).
 38. A method ofdecreasing the level of SRBP1c in the liver of a subject comprisingadministering to the subject vitamin A or an agonist of retinoic acidreceptor-beta (RARβ).
 39. A method of treating or preventing a kidneydisease in a subject comprising administering to said subject vitamin Aor an agonist of retinoic acid receptor-beta (RARβ).
 40. The method ofclaim 39, wherein said kidney disease is associated with obesity. 41.The method of claim 39, wherein said kidney disease is associated with ahigh fat diet.
 42. The method of claim 39, wherein said kidney diseaseis kidney fibrosis.
 43. The method of claim 39, wherein said kidneydisease is a chronic kidney disease.
 44. The method of claim 39, whereinsaid kidney disease is associated with a pancreatic disease.
 45. Themethod of claim 39, wherein said kidney disease is associated with aliver disease.
 46. The method of claim 39, wherein said kidney diseaseis associated with reduced vitamin A level in the kidney.
 47. A methodof increasing the level of lethicin:retinol acyltransferase (LRAT) inthe kidney of a subject comprising administering to said subject vitaminA or an agonist of retinoic acid receptor-beta (RARβ).
 48. A method oftreating or preventing a disease associated with an organ-specificvitamin A deficiency in a subject comprising administering to saidsubject vitamin A or an agonist of retinoic acid receptor-beta (RARβ).49. The method of claim 48, wherein said organ-specific vitamin Adeficiency is associated with obesity.
 50. The method of claim 48,wherein said organ-specific vitamin A deficiency is associated with ahigh fat diet.
 51. The method of claim 48, wherein said subject has anormal serum level of vitamin A.
 52. The method of claim 48, whereinsaid subject has a normal serum level of retinyl esters.
 53. The methodof claim 48, wherein said organ is pancreas, liver, kidney or testes.54. A method of treating or preventing fibrosis in a subject comprisingadministering to said subject an agonist of retinoic acid receptor-beta(RARβ).
 55. A method of decreasing the accumulation of fat in a subjectcomprising administering to said subject an agonist of retinoic acidreceptor-beta (RARβ).
 56. The method of claim 54, wherein said fibrosisis in pancreas, liver, kidney or testes.
 57. The method of claim 55,wherein said accumulation of fat is in pancreas, liver, kidney ortestes.
 58. The method according to any one of claims 1 to 58, whereinsaid vitamin A or agonist of retinoic acid receptor-beta (RARβ) isadministered three times daily.
 59. The method according to any one ofclaims 1 to 58, wherein said vitamin A or agonist of retinoic acidreceptor-beta (RARβ) is administered at an amount from about 30 mg toabout 200 mg per day.
 60. The method of claim 59, wherein said agonistis administered at an amount from about 50 mg to about 150 mg per day.61. The method of claim 59, wherein said agonist is administered at anamount from about 50 mg to about 100 mg per day.
 62. The method of claim59, wherein said agonist is administered at an amount from about 100 mgto about 150 mg per day.
 63. The method according to any one of claims 1to 58, wherein said vitamin A or agonist of retinoic acid receptor-beta(RARβ) is administered orally.
 64. The method according to any one ofclaims 1 to 58, wherein said vitamin A or agonist of retinoic acidreceptor-beta (RARβ) is administered intravenously or subcutaneously.65. The method according to any one of claims 1 to 58, wherein saidagonist of retinoic acid receptor-beta (RARβ) does not elevate serumtriglyceride in said subject.
 66. The method according to any one ofclaims 1 to 58, wherein said agonist of retinoic acid receptor-beta(RARβ) does not increase cardiovascular risk in said subject.
 67. Themethod according to any one of claims 1 to 58, wherein a therapeuticeffective amount of said vitamin A or agonist of RARβ is administered.68. The method according to any one of claims 1 to 58, comprising bothvitamin A and an agonist of RARβ.
 69. The method according to any one ofclaims 1 to 58, wherein said agonist is a highly specific RARβ agonist.70. The method according to any one of claims 1 to 58, wherein saidagonist is AC261066.
 71. The method according to any one of claims 1 to58, wherein said agonist is AC55649.
 72. A pharmaceutical compositioncomprising vitamin A or an agonist of retinoic acid receptor-beta (RARβ)or a pharmaceutically acceptable salt thereof at an amount from about 10mg to about 60 mg.
 73. The pharmaceutical composition of claim 72,wherein said amount of vitamin A or agonist is from 15 mg to about 50mg.
 74. The pharmaceutical composition of claim 72, wherein said amountof the vitamin A or agonist is from 15 mg to about 35 mg.
 75. Thepharmaceutical composition of claim 72, wherein said amount of thevitamin A or agonist is from about 35 mg to about 50 mg.
 76. Thepharmaceutical composition of claim 72, wherein said amount of thevitamin A or agonist is from about 30 mg to about 200 mg.
 77. Thepharmaceutical composition of claim 72, wherein said amount of thevitamin A or agonist is from about 50 mg to about 150 mg.
 78. Thepharmaceutical composition of claim 72, wherein said amount of thevitamin A or agonist is from about 50 mg to about 100 mg.
 79. Thepharmaceutical composition of claim 72, wherein said amount of thevitamin A or agonist is from about 100 mg to about 150 mg.
 80. Apharmaceutical composition comprising vitamin A or an agonist ofretinoic acid receptor-beta (RARβ) or a pharmaceutically acceptable saltthereof at a concentration from about 0.1 mg to about 10 mg per 100 ml.81. The pharmaceutical composition of claim 72, wherein saidconcentration is from about 0.5 mg to about 5 mg per 100 ml.
 82. Thepharmaceutical composition of claim 72, wherein said concentration isfrom about 1 mg to about 2.5 mg per 100 ml.
 83. The pharmaceuticalcomposition of claim 72, comprising both vitamin A and an agonist ofRARβ.
 84. The pharmaceutical composition according to any one of claims72 to 83, wherein said agonist is a highly specific RARβ agonist. 85.The pharmaceutical composition according to army one of claims 72 to 83,wherein said agonist is AC261066.
 86. The pharmaceutical compositionaccording to any one of claims 72 to 83, wherein said agonist isAC55649.